The present invention relates to fibroblast growth factor (FGF) binding and FGF receptor (FGFR) activation. More particularly, the invention relates to a method of identifying compounds that interact with FGF and modulate its activity such as, for example, activators and inhibitors.
(Note: Literature references on the following background information and on conventional test methods and laboratory procedures well known to the ordinary person skilled in the art, and such other state-of-the-art techniques as used herein, are indicated in parentheses and appended at the end of the specification.)
FGFs regulate a diverse range of physiologic processes such as cell growth and differentiation and pathologic processes involving angiogenesis, wound healing and cancer (1).
FGFs utilize a dual receptor system to activate signal transduction pathways (2-5). The primary component of this system is a family of signal-transducing FGF receptors (FGFRs) that contain an extracellular ligand-binding domain and an intracellular tyrosine kinase domain (1).
The second component of this receptor system consists of heparan sulfate (HS) proteoglycans or related heparin-like molecules which are required in order for FGF to bind to and activate the FGFR (3,4).
Heparin/HS may interact directly with the FGFR linking it to FGF (6). Furthermore, heparin/HS can facilitate the oligomerization of two or more FGF molecules, which may be important for receptor dimerization and activation (3). There are no pharmacologic agents that were previously known to modulate the activity of FGFs.
Heparin/HS is a heterogeneously sulfated glycosaminoglycan that consists of a repeating disaccharide unit of hexuronic acid and D-glucosamine. It has been previously reported that, at a minimum, highly sulfated octa- (3) or decasaccharide (7) fragments derived from heparin are required for FGF to bind to the FGFR. However, preparation of these heparin fragments produces mixtures of isomers and chemically modifies the oligosaccharide ends (8). Furthermore, size-fractionated heparin may contain individual molecules with distinct biological properties.
Accordingly, it would be desirable to determine the mechanism by which heparin/HS activates FGF and, further, to identify compounds that interact with FGF and modulate its activity.
In U. S. Pat. No. 5,270,197, various systems are described for assaying the ability of a substance to bind to a high-affinity heparin-binding growth factor (HBGF) receptor, e.g. an FGF receptor. The disclosure of said patent is incorporated herein by reference.